1. Field of the Invention
In one of its aspects, the present invention relates to an extract of a species of Nerium, particularly Nerium Oleander, and to a method for production thereof. In another of its aspects, the present invention relates to a pharmaceutical composition comprising an extract of a species of Nerium, particularly Nerium Oleander, and to a method for production thereof. The pharmaceutical composition is useful, inter alia, in the treatment of the cell-proliferative and immune deficient diseases in mammals, including cancer and AIDS.
2. Description of the Prior Art
A variety of herbal and plant extracts or preparations are available for mankind for treating any number of diseases affecting the human body [1, 4]. U.S. Pat. No. 4,986,895 [Grossman et al.] teaches the use of water soluble plant extracts in the treatment of virus skin infections. U.S. Pat. No. 5,178,865 [Ho et al.] teaches the use of Chinese herbal extracts in the treatment of HIV related disease in vitro. U.S. Pat. No. 5,482,711 [Medenica] teaches the use of the extract of the plant Nigella Sativa in the treatment of cancer, viral diseases and protection from side effects of chemotherapy. Many cell anti-proliferative agents are natural products of fungi, plants and marine animals, and these materials are likely to be the primary sources for anti-proliferative agents for the future. Examples of this class of agents include taxol, vincristine and camptothecin.
U.S. Pat. No. 5,135,745 [Ozel, 20], the contents of which are hereby incorporated by reference, teaches the use of the extract of the plant Nerium Oleander for the treatment of cell proliferation diseases in animals and humans. Ozel [20]teaches preparing the water extract of the plant Nerium Oleander and administering the extract to human subjects in order to ameliorate cell-proliferative diseases such as cancer. As used throughout this specification, the term xe2x80x9ccell-proliferative diseasexe2x80x9d is intended to mean malignant as well as non-malignant cell populations which often appear morphologically to differ from the surrounding tissue. Ozel [20] teaches a screen to be used in determining whether a particular patient would be a suitable candidate to receive a therapeutic regimen of the extract. Specifically, Ozel [20] teaches initial adminstration of an injectable form of the extract following by observation to detect the onset of fever (from about 38xc2x0 C. to about 41xc2x0 C.) in the patient. If the fever develops, the patient is deemed to be a suitable candidate for receiving a therapeutic regimen of the extract. Thus, Ozel [20] teaches a nexus between efficacy of the extract and the onset of a fever in the patient during the screening. In other words, Ozel teaches that a patient who fails to develop a fever during the screening will not be a suitable candidate for receiving a therapeutic regimen of the extract.
The plant oleander is a well-known ornamental plant with leathery evergreen leaves and handsome clusters of red or pink or white flowers. The plant originates from the Mediterranean region and is indigenous to the Indo-Pakistan subcontinent. The plant grows as a weed in the southern part of Texas. In the Mediterranean region, the plant previously has been used for a variety of medicinal purposes. For example: (i) the macerated leaves have been used to relieve itchiness and help prevent hair from falling out, (ii) fresh leaves have been applied to treat tumors, (iii) the decoction of leaves and bark has been used to treat syphillis, and (iv) the decoction of leaves has been used as a gargle to strengthen the teeth and gums and as a nose drop for children [1-4].
Oleander is one of the digitalis-like plants. The plant has certain toxic properties due to the presence of digitoxin like steroidal glycosides. It is estimated that as many as 100 chemical substances are present in various parts of the Oleander plant. Various of the compounds that have been identified in Nerium Oleander set out in Table 1.
Ozel describes a procedure for the preparation of Nerium Oleander Extract (NOE) in water [20, 21]. The specific extraction of the plant Nerium Oleander taught by Ozel [20, 21] involves, cooking the leaves and stems of the plant in water for 2-3 hours and filtering off the residues. Some of the chemical constituents have been separated from the aqueous extract and have been analyzed [22]. The extract has been found to comprise several polysaccharides with very potent immune stimulating properties. The various polysaccharides identified in the aqueous NOE set out in Table 2. These polysaccharides can be mixed with various pharaceutically acceptable carriers to form injectables, capsules, tablets and various other administrative forms [22].
While the extract taught by Ozel [20] is a significant advance in the art of treatment of cell-proliferative diseases in humans, there is still room for improvement. Specifically, as described above, Ozel [20] teaches that only patients who develop a fever during the screening procedure are suitable canditates to receive a therapeutic regimen of the extract. Further, there are technical problems in producing the extract in a commercially suitable form for parenteral administration using the process taught by Ozel [20]. In particular, there is a problem regarding the stability, sterility and endotoxin level of the extract during extended periods of use on human subjects. The aqueous NOE extract described in Ozel [20] is relatively unstable at room temperature over any significant period of time. In particular, the aqueous extract described in Ozel [20], loses its potency when stored at ambient temperatures.
Accordingly, it would be desirable to have an improved extract of the Nerium species which could be used with patients who do not develop a fever during initial screening. It would also be desirable if the extract itself had improved stability and could be used to produce a formulation of improved stability.
It is an object of the present invention to provide a novel extract of a species of Nerium which obviates or mitigates at least one of the above-mentioned disadvantages of the prior art.
It is another object of the present invention to provide a novel process for producing an extract of a species of Nerium.
It is yet another object of the present invention to provide a novel pharmaceutical composition which obviates or mitigates at least one of the above-mentioned disadvantages of the prior art.
It is yet another object of the present invention to provide a novel process for producing a pharmaceutical composition.
Accordingly, in one of its aspects, the present invention provides a substantially sterile extract of Nerium species.
In another of its aspects, the present invention provides a method for producing a substantially sterile extract of Nerium species, the method comprising the steps of:
(i) dispersing plant matter of the Nerium species in water;
(ii) heating the dispersed plant matter for at least about 1.0 hour;
(iii) separating the heated water from the plant matter to produce a crude extract; and
(iv) sterilizing the crude extract to produce the substantially sterile extract of Nerium species.
In yet another of its aspects, the present invention provides a pharmaceutical composition comprising a substantially sterile extract of Nerium species, together with at least one pharmaceutically acceptable excipient therefor.
In yet another of its aspects, the present invention provides a process for producing a pharmaceutical composition comprising the steps of:
(i) mixing a substantially sterile extract of Nerium species with at least one pharmaceutically acceptable excipient and water;
(ii) drying the mixture produced in Step (i).
In yet another of its aspects, the present invention provides a novel pharmaceutical composition for the treatment of cell-proliferative and immune deficient diseases in mammals, including cancer and AIDS, respectively. The pharmaceutical composition comprises a therapeutically effective amount of a substantially sterile extract from a species of Nerium, preferably made according to the process described below.
In yet another of its aspects, the present invention provides a novel pharmaceutical composition for pain remediation in mammals. The pharmaceutical composition comprises a therapeutically effective amount of a substantially sterile extract from a species of Nerium, preferably made according to the process described below.
While specific reference will be made in this specification to Nerium oleander extract (NOE), those of skill in the art will appreciate that the present invention may be applied to various species of the the Nerium genus. For the sole purpose of convenience and ease of understanding, reference is made in the illustrative embodiments to NOE.
In a further aspect of the present invention there is provided a dried NOE powder suitable for use as a pharmaceutical composition. This powder is stable for at least approximately 3 to 5 years. The NOE powder may be freeze-dried, dried by heating and evaporation or spray-dried. The NOE powder of known quantity may be mixed with required excipients and water to form an aqueous solution comprising NOE which in turn is dried to form a powdered pharmaceutical composition which is also stable for at least approximately 3 to 5 years. The powdered pharmaceutical composition can then be quickly reconstituted with water prior to administration to form a clear solution. This solution is stable for a period of approximately one month.
In yet another of its aspects, the present invention provides a method for making a freeze-dried pharmaceutical composition comprising a therapeutically effective amount of NOE which is stable for at least 3 to 5 years. The method for making a freeze-dried pharmaceutical composition comprising a therapeutically effective amount of NOE comprises the steps of.
(i) mixing a NOE powder with the desired excipients and dissolving in water; maintaining the pH of the solution in the range of from about 5 to about 8, preferably from about 6 to about 7;
(ii) filtering the solution with filter having an average pore size of less than about 1.0 xcexcm for sterilization; and
(iii) freeze-drying the solution under sterile conditions.
Preferably, freeze-drying is conducted in individual enclosures (e.g., vials) which are then sealed under the original vacuum in the freeze-drying compartment and can be reconstituted for use on human subjects whenever necessary. Thus, the present invention provides a means to produce a pharmaceutical composition containing a required amount of NOE powder.
In yet another of its aspects, the present invention provides a pharmaceutical topical cream comprising an amount of an NOE powder therapeutically effective for treating cell-proliferative diseases in mammals, together with a suitable exciptient therefor. Preferably, the excipient comprises an oil-in-water, semi-solid dosage form. A particularly preferred embodiment of such an excipient is discussed hereinbelow.
In a further aspect of the present invention there is provided a method of treating a mammal using a suitable pharmaceutical composition as described herein.
A number of different excipients may be used in the compositions of the present invention. These will be discussed in more detail hereinbelow.
In yet another of its aspects, the present invention provides a pharmaceutically acceptable parenteral preparation of NOE suitable especially for intramuscular or subcutaneous injections in human or animal subjects. The NOE product manufactured using the present invention is highly suitable for commercial shipping, handling and storing under ambient temperature. The product manufactured based on the present invention is stable at ambient temperature for at least up to 3 to 5 years. Thus, this aspect of the present invention may be used to manufacture commercial quantities of vials containing NOE.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying Tables.
Thus, an aspect of the present invention relates to a substantially sterile extract of Nerium species. As used throughout this specification, the term xe2x80x9csubstantially sterilexe2x80x9d, when used in conjunction with an extract of Nerium species, is intended to mean an extract which meets the requirements under the xe2x80x9cSterility Testsxe2x80x9d as described in the United States Pharmacopeia, The National Formulary, USP 23, NF 18, USP, pgs. 1686-1690, Rockville, Md. (1995), the contents of which are hereby incorporated by reference. In this regard, it should be noted that the extract taught by Ozel [20] does not meet these requirements.
Preferably, the concentration of bacterial endotoxins in the present extract is less than about 300 units/mL, preferably in the range of from about 20 units/mL to about 150 units/mL, more preferably in the range of from about 20 units/mL to about 120 units/mL, even more preferably in the range of from about 20 units/mL to about 100 units/mL, even more preferably in the range of from about 20 units/mL to about 80 units/mL, most preferably from about 20 units/mL to about 50 units/mL.
The determination of the concentration of bacterial endotoxins in the extract is within the purview of a person of ordinary skill in the art. Preferably, the concentration of bacterial endotoxins in the extract may be determined by performing a conventional Limulus Amebocyte Lysate (LAL) Test as set out in the United States Pharmacopeia, The National Formulary, USP 23, NF18, USP, pgs. 1696-1697, Rockville, Md. (1995), the contents of which are hereby incorporated by reference.
The species of Nerium useful to produce the present extract is not particularly restricted. For example, three Nerium species, Nerium Indicum, Nerium Oleander and Nerium Odorum, may be found in various regions from the Mediterranean to Japan. Preferably, the species of Nerium used in the present invention is Nerium Oleander, also known as pink oleander and belonging to the Apocynceae family. As is known, pink oleander is a shrub that grows about 8-12 feet tall with leathery long leaves about 9 inches long and 1 inch wide. The shrub flowers in red, pink, white or cream colored clusters from spring to fall. All parts of the plant, including the leaves, flowers and twigs contain cardiac glycosides such as oleandrin, neriin, folinerin, digitoxigenin and nerigoside.
The physical form of the substantially sterile extract of the present invention is not particularly restricted. In one embodiment, the extract is in a liquid, preferably aqueous, form. In another embodiment, the extract is in a solid, preferably powedered, form. If the extract is in a powdered form, preferably, it is prepared by drying a liquid composition comprising the extract.
The specific drying technique is not particularly restricted provided that it does not adversely affect the active constituents of the extract. Non-limiting examples of useful drying techniques include: lyophilization (freeze-drying), spray-drying and evaporation of the solution under reduced pressure.
The lyophilization method is the preferred method for the preparation of NOE powder because the chemical entities, including the polysaccharides, present in the extract solution are well preserved with respect to the molecular structure which is essentially not altered during the drying process under sub-zero conditions. The preparation of the NOE powder by lyophillization is described in detail in Example 1 hereinbelow. In the lyophillization process, the NOE solution is normally frozen rapidly in stainless steel metal tray containers placed in the shelf of the freeze-dryer which is kept at a temperature in the range of from about xe2x88x9235xc2x0 C. and about xe2x88x9240xc2x0 C. The frozen solution is then lyophilized and a fluffy dry powder results from the removal of water by freeze-drying. This NOE powder may be stored for extended periods under conditions that reduce chemical degradation of the constituents or the absorption of moisture by the extract solution. Examples of such conditions include powder sealed under an atmosphere of dry, inert gas (such as argon, nitrogen, etc.) and storage below zero degree centigrade. Further, the lyophilized NOE powder can be readily dissolved in pyrogen-free deionized water or any other form of water suitable for injection to produce the extract without any significant loss of its biological potency.
The NOE powder can also be prepared by a conventional spray-drying technique. Spray drying is a process of converting a liquid into a powder by spraying the liquid into a hot drying gaseous medium. This process constitutes: (1) generation of liquid aerosol droplets, (2) evaporation of solvent from these droplets resulting in solution supersaturation, and (3) nucleation and precipitation of the supersaturated solution within the droplets.
The present substantially sterile extract of Nerium species may produced by a method comprising the steps of:
(i) dispersing plant matter of the Nerium species in water;
(ii) heating the dispersed plant matter for at least about 1.0 hours;
(iii) separating the heated water from the plant matter to produce a crude extract; and
(iv) sterilizing the crude extract to product the substantially sterile extract of Nerium species.
The production of the crude extract (i.e., Steps (i), (ii) and (iii)) is generally similar to the technique disclosed in U.S. Pat. No. 5,135,745 [Ozel, 20], the contents of which are hereby incorporated by reference.
Preferably, Step (ii) is conducted at a temperature of at least about 40xc2x0 C., more preferably in the range of from about 70xc2x0 C. to about 120xc2x0 C., even more preferably in the range of from about 80xc2x0 C. to about 110xc2x0 C., most preferably in the range of from about 90xc2x0 C. to about 100xc2x0 C.
Preferably, Step (ii) is conducted for a period in the range of from about 1 to about 24 hours, more preferably from about 1 to about 10 hours, most preferaby from about 1 to about 5 hours.
Step (iv) serves to convert the crude extract to a substantially sterile extract. This may be accomplished, for example, by filter sterilization of the crude extract using the procedure for sterile filtration set out in USP Monograph (USP 23 less than 71 greater than ), the contents of which are hereby incorporated by reference. Preferably, the curde extract is converted to a substantially sterile extract by passing the crude extract through a filter having an average porosity of less than about 1 xcexcm, more preferably less than about 0.8 xcexcm, even more preferably less than about 0.5 xcexcm, most preferably less than about 0.2 xcexcm.
During Step (iv), the concentration of endotoxins in the crude extract is reduced to a value less than about 300 units/mL, preferably in the range of from about 20 units/mL to about 150 units/mL, more preferably in the range of from about 20 units/mL about 120 units/mL, even more preferably in the range of from about 20 units/mL to to about 100 units/mL, even more preferably in the range of from about 20 units/mL to about 80 units/mL, most preferably from about 20 units/mL to about 50 units/mL.
In a preferred embodiment of the method, the crude extract is subject to a further heating step prior to Step (iv). The duration and temperature of this additional heating step may be selected from the preferred embodiments of the Step (ii) discussed hereinabove.
The product obtained by this method is a substantially sterile extract of Nerium species which, together with at least one pharmaceutically acceptable excipient, may be formulated into a pharmaceutical composition. While the active substances in the extract can be administered alone, it is generally preferred that they be administered with at least one pharmaceutically acceptable excipient, the latter selected on the basis of the chosen route of administration and standard pharmaceutical practice. The term xe2x80x9cpharmaceutically acceptable excipientxe2x80x9d is intended to mean an ingredient used in a pharmaceutical preparation which does not function as an active agent.
Thus, the present pharmaceutical composition may be formulated oral, topical, rectal, parenteral, local, inhalant, topical or intracerebral administration. In an embodiment of the invention, the substances are administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using forms of transdermal skin patches known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will be continuous rather than intermittent throughout the dosage regimen. The substances can also be administered by way of controlled or slow release capsule system and other drug delivery technologies.
Thus, the present substantially sterile extract of Nerium species is typically administered in admixture with suitable pharmaceutical diluents or carriers suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, consistent with conventional pharmaceutical practices.
For example, for oral administration in the form of a tablet or capsule, the active substances can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral active substances can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the dosage form if desired or necessary. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Suitable lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Examples of disintegrators include starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
Gelatin capsules may contain the active substance and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar carriers and diluents may be used to make compressed tablets. Tablets and capsules can be manufactured as sustained release products to provide for continuous release of active ingredients over a period of time. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and flavouring agents to increase patient acceptance.
Water, a suitable oil, saline, aqueous dextrose, and related sugar solutions and glycols such as propylene glycol or polyethylene glycols, may be used as carriers for parenteral solutions. Such solutions also preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Suitable stabilizing agents include antioxidizing agents such as sodium bisulfate, sodium sulfite, or ascorbic acid, either alone or combined, citric acid and its salts and sodium EDTA. Parenteral solutions may also contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
The substantially sterile extract of the invention can also be administered topicially, for example, in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
A particuarly preferred form of topical administration of the present substantially sterile extract of the invention is via a cream composition. Preferably, the composition is formulated in an oil-in-water, semi-solid dosage form. In this case, the active ingredient (i.e., the substantially sterile extract of Nerium species), may be formulated in a moisturizing base that has beneficial effect on its own.
A preferred cream-based formulation comprises: (i) emulsifying, defoaming and thickening agents, (ii) an antioxidant, (iii) an emollient humectant, (iv) preservatives/antimicorbial agents (optional), and (v) a diluent (optional).
Non-limiting examples of suitable emulsifying, defoaming and thickening agents may be selected from the group comprising Meezawax(trademark), glyceryl monostearate, cetyl alcohol, polyoxy 50 stearate, Tween(trademark) 20, petrolatum lanolin alcohol, liquid lanolin and mixtures thereof. Preferably, these agents may be used in an amount of up to about 40%, preferably in the the range of from about 10% to about 40%, by weight of the composition. A non-limiting example of a suitable antioxidant is vitamin E acetate. Preferably, the antioxidant is used in an amount of up to about 5%, preferably in the the range of from about 1% to about 3%, by weight of the composition.
A non-limiting example of a suitable emollient humectant is glycerin. Preferably, the emollient humectant is used in an amount of up to about 15%, preferably in the the range of from about 5% to about 10%, by weight of the composition.
The emulsifying, defoaming and thickening agents, antioxidant and emollient humectant make up a cream base which facilitates application of the active ingredient to the skin.
It is known in the art to add preservatives/antimicorbial agents to cream formulations to improve the shelf life of the formulation. Non-limiting examples of suitable such agents may be selected from the group comprising methyl paraben, propyl paraben, potassium sorbate and mixtures thereof. If present, it is preferred to use methyl paraben in an amount of up to about 0.3%, preferably in the the range of from about 0.05% to about 0.2%, by weight of the composition. If present, it is preferred to use propyl paraben in an amount of up to about 0.03%, preferably in the the range of from about 0.005% to about 0.02%, by weight of the composition. If present, it is preferred to use potassium sorbate in an amount of up to about 0.3%, preferably in the the range of from about 0.01% to about 0.2%, by weight of the composition.
A non-limiting example of a suitable diluent is de-ionized water which also has some moisturizing properties.
The substantially sterile extract of the invention may also be coupled with soluble polymers which are targetable drug carriers. Examples of such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. The extract may also be coupled to biodegradable polymers useful in achieving controlled release of a drug. Suitable polymers include polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels. The extract may also be affixed to rigid polymers and other structures such as fullerenes or Buckeyballs.
Suitable pharmaceutical carriers and methods of preparing pharmaceutical dosage forms are described in Remington""s Pharmaceutical Sciences (Mack Publishing Company), the British Pharmacopeia, the Japanese Pharmacopeia, the United States Pharmacopeia XXII and the National Formulary XVII and supplements thereto, each of which is standard reference text in this field, and the contents of each of which are hereby incorporated by reference.
A preferred antioxidant excipient is a substance, which is added to a pharmaceutical preparation, inhibits oxidation and deterioration of the pharmaceutical preparation by oxidative processes. Such processes include the development of rancidity in oils and fats or the inactivation of some medicinals in the environment of their dosage forms. Non-limiting examples of antioxidants suitable for injection include ascorbic acid, sodium ascorbate, sodium bisulfate, sodium metabisulfate, DL-alpha-Tocopherol, monothioglycerol and mixtures thereof. In addition, there are other chemical agents available for use as antioxidants in a pharmaceutical preparation.
A preferred preservative excipient is a substance that prevents or inhibits microbial growth and may be added to a pharmaceutical preparation for this purpose to avoid consequent spoilage of the preparations by microorganisms. Non-limiting examples of suitable anti-microbial preservatives include methylparaben, methylparaben sodium, propyl paraben, propyl paraben sodium, ethylparaben, butylparaben, benzoic acid, sodium benzoate, potassium benzoate, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, benzalkonium chloride, benzthonium chloride and mixtures thereof.
The present pharmaceutical composition may contain, inter alia, solubilizer excipients or surfactant excipients suitable for injection. Non-limiting examples thereof include various Tween(trademark) products such as Tween(trademark) 80, Tween(trademark) 20, Tween(trademark) 40, Tween(trademark) 60, lecithin, sodium lauryl sulfate and mixtures thereof.
The present pharmaceutical composition may be produced by a process comprising comprising the steps of:
(i) mixing a substantially sterile extract of Nerium species with at least one pharmaceutically acceptable excipient and water;
(ii) drying the mixture produced in Step (i).
Step (i) may be accomplished by dissolving a previously dried sample of substantially sterile extract of Nerium species in a suitable solvent and adding the excipient(s) thereto. Alternatively, it may be possible to use the product of the process to produce the extract direcly in Step (i) of this process. The relative amount of extract used in Step (i) may vary depending on the amount of excipients added and the type of cell-proliferative diseases to be treated with the pharmaceutical composition. Preferably, the extract is used in the form of NOE powder, more preferably in an amount in the range of from about 0.01 mg/mL to about 100 mg/mL, most preferably in the range of from about 1 mg/mL to about 30 mg/mL. Antioxidants, preservatives and other any other desirable excipients may be added with or subsequent to adding the NOE powder.
The resulting solution may be sterilized using any of the known methods appropriate to preserving the active constituents. The preferred method of sterilizing the resulting solution is by filtering through a micron filter as discussed hereinabove using sterile equipment and techniques. Alternatively, the components of the pharmaceutical composition may be sterilized by any of the known methods appropriate to preserving the active compound prior to mixing in water and may be mixed using sterile equipment and technique. The sterile filtering method is preferred for the pharmaceutical composition of the present invention because the active constituents will not be destroyed by the process. For example, the solution may be dispensed in a sterile container (e.g., a vial) under sterile conditions, lyophilized and capped under original vacuum. Prior to use, the lyophilized pharmaceutical preparation can be reconstituted for injection using water suitable for injection.
The pharmaceutical composition of the present invention is relatively non-toxic. For example, the LD50 value of the pharmaceutical composition containing 15 mg/mL by intravenous injection in mice is more than 4 mL/kg. It is well documented in the scientific literature that polysaccharides derived from plants, fungi, bacteria, algae and marine weeds a have wide range of biological activities, including, anti-tumor [24-28], immunological [29-32], anti-complimentary [33-37], anti-inflammatory, anti-coagulant, hypoglycemic [41-43] and anti-viral [44-45] activities. While not wishing to be bound by any specific theory or mode of action, the anti cell-proliferative activities of the pharmaceutical composition of the present invention are believed to be attributable to the polysaccharides present in the solution and the biological effect of these polysaccharides is believed to be primarily due to their potent immune stimulating properties.
Immune stimulation occurs as a result of injection of substances which themselves have only weak antigenic effects, but are nevertheless able to induce the body""s own defense mechanisms in a non-specific or even specific manner. Among other things, these substances generally influence the proliferation of the immunocompetant cells, but they do not leave behind any memory reaction. This means that the primary targets of the action of the immunostimulating substances are the macrophages and granulocytes, as well as T- and B-lymphocytes. The effect of the immunostimulants may be direct or indirect, e.g., via the complement system or the lymphocytes, via the production of interferon or lysosomal enzymes (e.g., lymphokines, colony stimulating factor and others), as well as via the increase in macrophagocytosis and microphagocytosis.
Certain anionic polysaccharides [46, 47], such as dextran sulphate and pustulan sulphate stimulate cell-mediated T-cell dependent immune responses without stimulating antibody mediated immune responses that are B-cell dependent. On the other hand, unmodified polysaccharides stimulate only B-cells and certain other polysaccharides are known to stimulate both T-cell and B-cell responses under certain conditions. At present, the inventors are not aware of a specific test method to measure the stimulating effect of a given compound. Instead, the stimulating effect is measured in an indirect way.
The polysaccharides present in the present pharmaceutical composition (Table 2 above), namely, PS-I, PS-II, PS-III and PS-IV, have been shown to stimulate the immune system by measuring the effect of these polysaccharides on the function and capability of mononucleated systems as well as the ability to stimulate T- and B-lymphocytes. For example, the polysaccharide PS-I is shown to produce 256 Unit/mL of Tumor Necrosis Factor (TNF) at a concentration of 50 mcg/mL while a mixture of PS-II, PS-III and PS-IV is shown to produce 1000 Unit/mL of TNF at a concentration of 3 mcg/mL. In addition, these polysaccharides have been shown to stimulate the lymphocyte proliferation and phagocyte values as described in the examples below.
While the immune stimulating properties of the polysaccharides present in the substantially sterile extract of the present invention has been established, it is believed that the extract may also contain other molecules both small (i.e., less than 1000 Dalton) and large (i.e., greater than 1000 Dalton) in size with immune stimulating and other biological properties. Thus, the properties of amelioration of the cell-proliferative diseases by the pharmaceutical composition of the present invention is believed not to be limited to the presence of the identified polysaccharides PS-I, PS-II, PS-III and PS-IV in the composition.
Thus, in a preferred embodiment, the present invention provides for the manufacture of a pharmaceutical composition containing NOE powder suitable for paranteral administration to human and animal subjects. In particular, NOE powder and the lyophilized pharmaceutical compositions described in this invention have been shown to be stable up to three years and can be conveniently stored at ambient temperature with minimal or no loss in activity. Furthermore, in a preferred embodiment, the lyophilized vials containing the pharmaceutical composition can be shipped to customers without loosing any biological activities due to ambient temperature and humidity changes. Also, the process of the present invention can be used successfully to scale-up the production of the pharmaceutical composition described here.
Another aspect of the present invention is directed to therapeutically inhibiting cell-proliferative diseases such as cancer and and immune deficient diseases such as AIDS. Accordingly, human patients suspected of having a malignant cell-proliferative disease or viral disease, such as AIDS, are initially given a low daily dose of 0.3 mL of the pharmaceutical composition of NOE of the present invention intramuscularly on the first day and the dosage is increased to 0.5 mL within the next seven days. After seven days, the patients are placed on an initial therapeutic regimen utilizing NOE. This therapeutic regimen is performed typically for once a day for a period of three months to one year depending upon the severity of the disease. Following completion of a normal therapeutic regimen, a patient is then placed on a maintenance therapeutic regimen for a period of time ranging from about six months to about three years. During this period NOE is administered once every two days for a period of about six months. After this treatment, the patient is given NOE once a week for up to three years.
In order to illustrate the anti cell-proliferative and pain remediation activities of the present pharmaceutical formulation we have provided five case reports in Examples 8-16 below of human subjects who have been treated with the pharmaceutical composition of the present invention.
The pharmaceutical composition according to the present invention offers several advantages over the formulations disclosed in U.S. Pat. No. 5,135,745 [Ozel, 20]. Specifically, Ozel teaches a screening technique in which potential patients are assessed for the onset of fever as the benchmark for treatment. In other words, Ozel teaches that only those patients who develop an initial fever are suitable for receiving a therapeutic regimen of the formulation. In contrast, the present inventors have surprisingly and unexpectedly discovered that the onset of fever is not necessarily linked to efficacy of the present substantially sterile extract of Nerium species. Specifically, after taking the present formulations intramuscularly the patients do not produce fever as in the case of the previous formulation. Thus, whereas the original Ozel formulation teaches awaing from efficacy in patients who do not exhibit the onset of fever, the present pharmaceutical composition is effective, regardless of whether fever occurs in the patient. This renders the present pharmaceutical compositions useful in a wide variety of patients and in a more patient-friendly manner. Further, the present substantially sterile extract of Nerium species is stable for several years unlike the original crude extract of Ozel [20].
A number of excipients may be appropriate for use in the formulations, which comprise the composition according to the present invention. The inclusion of excipients and the optimization of their concentrations for their characteristics will be understood by those ordinarily skilled in the art.
Embodiments of the invention will now be illustrated with reference to the following Examples. The Examples are intended to be merely illustrative of the invention and should not be used to limit or construe the invention.